Evaluation of in vitro effects of different agents on hydatid cyst scolices

Objective: Hydatid cyst, is a zoonotic disease primarily caused by Echinococcus granulosus, with humans being accidental hosts. The liver is the most commonly affected organ in hydatid cyst disease. Symptoms are usually nonspecific until the cyst reaches a certain size, making diagnosis challenging. Treatment varies depending on factors such as cyst size, location, radiological stage, and the presence of complications. Treatment options include observation, medical treatment, percutaneous interventions, and surgery. Echinococcus granulosus is widely distributed geographically and is endemic also in Turkey. Recurrence rates may vary with different treatment modalities. Albendazole is commonly used in medical treatment today. In surgical interventions and percutaneous procedures, scolicidal agents are injected into the cyst to prevent spillage by preserving the peritoneal cavity and ensuring sterilization within the cyst, aiming to reduce recurrence rates. While hypertonic saline and ethanol are commonly used for injection, other agents with proven scolicidal activity are also available. However, agents used for intra-cystic injections may have undesirable side effects such as hypernatremia, sclerosing cholangitis, biliary strictures, peritonitis, and toxicity. Therefore, the search for new scolicidal agents continues. An ideal scolicidal agent for intra-cystic injections should have high scolicidal activity, rapid effectiveness, and no adverse effects. In this context, plant extracts and their active compounds are also being evaluated. Particularly, agents proven effective against malaria are considered to potentially be effective against Echinococcus species as well. Therefore, our aim was to investigate the scolicidal activity of Artemisia annua and Cinchona officinalis plant extracts, which have shown efficacy against malaria, and also to contribute to the search for scolicidal agents by studying the extracts of Camellia sinensis and Commiphora molmol, which have demonstrated effectiveness against other parasites. Materials and Methods: The contents of a large solitary hepatic cyst consistent with Gharbi type 3 hydatid cyst were aspirated and collected in a syringe during surgery from a patient. After opening the cyst roof, daughter cysts were collected without opening and were washed with isotonic solution to prepare them for the experiment. Daughter cysts prepared for the experiment were opened within the aspirated cyst fluid and then all cyst contents were collected in glass cylinders. After waiting for a certain period, protoscoleces settled at the bottom, and the supernatant remaining at the top was discarded. Then, protoscoleces were washed three times with phosphate buffered saline (PBS). Their viability was confirmed by their failure to be stained with eosin and their motility. In the examination, 200.000 protoscoleces/cc were counted, and a viability rate of over 95% was determined. Extracts of Cinchona officinalis, Camellia sinensis, Commiphora molmol, and Artemisia annua were prepared by us, and the Artemisia annua extract was obtained ready-made from an external source. Each plant extract was prepared in three different concentrations of 25 mg/ml, 12,5 mg/ml, and 6,25 mg/ml dissolved in dimethyl sulfoxide (DMSO). DMSO was used as the negative control group, and 20% hypertonic saline was used as the positive control group. 0,05 ml of each concentration of the agent to be tested was dispensed into wells of a well plate, and 0,05 ml of cyst fluid containing protoscoleces was added on top. The counts were performed by dropping 0,01% eosin solution onto slides together with each plant extract at 5, 15, and 25 minutes under a light microscope. Dead protoscoleces were stained red with eosin, while live ones remained unstained. Each experiment was repeated three times. The number of dead scoleces found under the light microscope was calculated as a percentage to all scoleces to determine the scolicidal activity. Results: Extracts of Camellia sinensis and Commiphora molmol showed no effective scolicidal activity even at the highest concentration used. The average scolicidal activity observed after 5, 15, and 25 minutes of using 25 mg/ml of Camellia sinensis extract was calculated as 46,15%, 58,83%, and 76,81% respectively. Similarly, 25 mg/ml of Commiphora molmol extract showed scolicidal activity at rates of 31,51%, 34,2%, and 45,25% after 5, 15, and 25 minutes respectively. The highest scolicidal activity was demonstrated by Cinchona officinalis extract. When used at a concentration of 25 mg/ml, it was observed to be consistently more effective than 20% hypertonic saline at all time intervals. At 5, 15, and 25 minutes, it showed an average scolicidal activity of 89,08%, 95,01%, and 100% respectively. The average scolicidal activity of 20% hypertonic saline was calculated as 62,8% at 5 minutes, 76,95% at 15 minutes, and 89,65% at 25 minutes. Artemisia annua extract reached its highest effectiveness at 25 minutes with an average scolicidal activity of 87,25% at a concentration of 25 mg/ml. The weakest effect was observed in the negative control group using DMSO, showing an average scolicidal activity of 15,78% at 25 minutes, making it by far the least effective agent. Conclusion: This in vitro study demonstrates that Cinchona officinalis and Artemisia annua could serve as alternatives as scolicidal agents in the treatment of hydatid cysts. Although no statistically significant difference was found between Cinchona officinalis and 20% hypertonic saline, 25 mg/ml of Cinchona officinalis extract was consistently more active than 20% hypertonic saline at all three time intervals and was the only agent to achieve 100% scolicidal efficacy at 25 minutes. In the search for new scolicidal agents, the agents we find effective need to be supported by new in vivo studies. Keywords: Hydatid cyst, cystic echinococcosis, liver cyst, in vitro, scolicidal agents, Cinchona officinalis, Artemisia annua, hypertonic saline, Commiphora molmol, Camellia sinensis